Diagnosis of Clostridium difficile infection using real-time PCR.

Clostridium difficile is known to cause antibiotic-associated diarrhea and pseudomembranous colitis. Toxinogenic strains of the bacterium produce toxins A (TcdA) and B (TcdB), which are associated with the pathogenicity. The standard methods for diagnosis of C. difficile infection include the cell cytotoxicity assay and the culture of a toxinogenic strain. Due to the long turnaround time of these methods, more rapid methods are preferred. Enzyme immunoassays are fast, but lack sensitivity. Therefore, real-time PCR methods have been developed. The real-time PCR described in this chapter detects tcdB, the gene coding for toxin B. Since toxin A-negative, toxin B-positive strains have been reported to cause disease as well, these strains can also be detected by this method which uses an automated STAR-MagnaPure method for the optimum isolation of DNA from feces. An internal control is included as well to control for inhibition of the PCR method.

The relation between order of acquisition, segmental frequency and function: the case of word-initial consonants in Dutch.

The impact of input frequency (IF) and functional load (FL) of segments in the ambient language on the acquisition order of word-initial consonants is investigated. Several definitions of IF/FL are compared and implemented. The impact of IF/FL and their components are computed using a longitudinal corpus of interactions between thirty Dutch-speaking children (age range: 0 ; 6-2 ; 0) and their primary caretaker(s). The corpus study reveals significant correlations between IF/FL and acquisition order. The highest predictive values are found for the token frequency of segments, and for FL computed on minimally different word types in child-directed speech. Although IF and FL significantly correlate, they do have a different impact on the order of acquisition of word-initial consonants. When the impact of IF is partialed out, FL still has a significant correlation with acquisition order. The reverse is not true, suggesting that the acquisition of word-initial consonants is mainly influenced by their discriminating function.

Consonant inventories in the spontaneous speech of young children: a bootstrapping procedure.

Consonant inventories are commonly drawn to assess the phonological acquisition of toddlers. However, the spontaneous speech data that are analysed often vary substantially in size and composition. Consequently, comparisons between children and across studies are fundamentally hampered. This study aims to examine the effect of sample size on the resulting consonant inventories. A spontaneous speech corpus of 30 Dutch-speaking 2-year-olds was used. The results indicate that in order to construct and compare inventories reliably, they should be drawn from speech samples that are equally large. A new consonant inventory procedure is introduced. The implementation of this procedure demonstrates considerably less variation in inventory size across children and word positions than reported previously. This finding has important implications for clinical studies that constructed and compared inventories of typically and atypically developing children without normalizing the sample size.

How to measure the onset of babbling reliably?

Various measures for identifying the onset of babbling have been proposed in the literature, but a formal definition of the exact procedure and a thorough validation of the sample size required for reliably establishing babbling onset is lacking. In this paper the reliability of five commonly used measures is assessed using a large longitudinal corpus of spontaneous speech from forty infants (age 0 ; 6-2 ; 0). In a first experiment it is shown that establishing the onset of babbling with reasonable (95%) confidence is impossible when the measures are computed only once, and when the number of vocalizations are not equal for all children at all ages. In addition, each measure requires a different minimal sample size. In the second experiment a robust procedure is proposed and formally defined that permits the identification of the onset of babbling with 95% confidence. The bootstrapping procedure involves extensive resampling and requires relatively few data.

Comparison of molecular typing methods applied to Clostridium difficile.

Since the 1980s the epidemiology of Clostridium difficile infection (CDI) has been investigated by the application of many different typing or fingerprinting methods. To study the epidemiology of CDI, a typing method with a high discriminatory power, typeability, and reproducibility is required. Molecular typing methods are generally regarded as having advantages over phenotypic methods in terms of the stability of genomic markers and providing greater levels of typeability. A growing number of molecular methods have been applied to C. difficile. For the early and rapid detection of outbreak situations, methods such as restriction enzyme analysis, arbitrary primed polymerase chain reaction (PCR), and PCR ribotyping are commonly used. For long-term epidemiology, multilocus sequence typing, multilocus variable number of tandem repeats analysis, and amplified fragment length polymorphism are of interest. Currently, the PCR-ribotyping method and the library of PCR ribotypes in Cardiff are the benchmarks to which most typing studies around the world are compared. Multilocus variable number of tandem repeats analysis is the most discriminative typing method and will contribute significantly to our understanding of the epidemiology of this important nosocomial pathogen.

Comparison of seven techniques for typing international epidemic strains of Clostridium difficile: restriction endonuclease analysis, pulsed-field gel electrophoresis, PCR-ribotyping, multilocus sequence typing, multilocus variable-number tandem-repeat analysis, amplified fragment length polymorphism, and surface layer protein A gene sequence typing.

Using 42 isolates contributed by laboratories in Canada, The Netherlands, the United Kingdom, and the United States, we compared the results of analyses done with seven Clostridium difficile typing techniques: multilocus variable-number tandem-repeat analysis (MLVA), amplified fragment length polymorphism (AFLP), surface layer protein A gene sequence typing (slpAST), PCR-ribotyping, restriction endonuclease analysis (REA), multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). We assessed the discriminating ability and typeability of each technique as well as the agreement among techniques in grouping isolates by allele profile A (AP-A) through AP-F, which are defined by toxinotype, the presence of the binary toxin gene, and deletion in the tcdC gene. We found that all isolates were typeable by all techniques and that discrimination index scores for the techniques tested ranged from 0.964 to 0.631 in the following order: MLVA, REA, PFGE, slpAST, PCR-ribotyping, MLST, and AFLP. All the techniques were able to distinguish the current epidemic strain of C. difficile (BI/027/NAP1) from other strains. All of the techniques showed multiple types for AP-A (toxinotype 0, binary toxin negative, and no tcdC gene deletion). REA, slpAST, MLST, and PCR-ribotyping all included AP-B (toxinotype III, binary toxin positive, and an 18-bp deletion in tcdC) in a single group that excluded other APs. PFGE, AFLP, and MLVA grouped two, one, and two different non-AP-B isolates, respectively, with their AP-B isolates. All techniques appear to be capable of detecting outbreak strains, but only REA and MLVA showed sufficient discrimination to distinguish strains from different outbreaks.

Typing and subtyping of Clostridium difficile isolates by using multiple-locus variable-number tandem-repeat analysis.

Using the genomic sequence of Clostridium difficile strain 630, we developed multiple-locus variable-number tandem-repeat analysis (MLVA) with automated fragment analysis and multicolored capillary electrophoresis as a typing method for C. difficile. All reference strains, representing 31 serogroups, 25 toxinotypes, and 7 known subtypes of PCR ribotype 001, could be discriminated from each other. Application of MLVA to 28 isolates from 7 outbreaks due to the emerging hypervirulent PCR ribotype 027-pulsed-field gel electrophoresis type NAP1 resulted in recognition of 13 clusters. Additionally, 29 toxin A-negative, toxin B-positive isolates belonging to PCR ribotype 017 from eight different countries revealed eight country-specific clusters. MLVA is a highly discriminatory genotyping method and a new tool for subtyping of newly emerging variants of C. difficile.

Evaluation of real-time PCR and conventional diagnostic methods for the detection of Clostridium difficile-associated diarrhoea in a prospective multicentre study.

In this prospective multicentre study, an enzyme-linked fluorescent assay (VIDAS CDA2; bioMérieux), an enzyme-linked assay [Premier Toxins A and B (PTAB); Meridian] and an in-house real-time PCR amplifying the tcdB gene were compared with the cell cytotoxicity assay used as the 'gold standard' for diagnosis of Clostridium difficile-associated diarrhoea (CDAD). Faecal samples from patients with a request for C. difficile diagnosis and samples from patients with diarrhoea hospitalized for at least 72 h were collected for 3 consecutive months from four university medical centres in The Netherlands. In total, 547 faecal samples were obtained from 450 patients. Of 540 samples available for all of the assays, 84 (15.6 %) showed a positive result in one or more assays. The cell cytotoxicity assay was positive in 31 samples (5.7 %) from 28 patients. A diagnosis of CDAD was not considered by the physician in 5 (23.8 %) of 21 patients with CDAD who were hospitalized for at least 72 h. Compared with the cell cytotoxicity assay, the sensitivity of VIDAS, PTAB and PCR was 83.9, 96.8 and 87.1 %, respectively. The specificity of VIDAS, PTAB and PCR was 97.1, 94.3 and 96.5 %, respectively. The positive and negative predictive values for VIDAS, PTAB and PCR were 63.4 and 99.0 %, 50.9 and 99.8 %, and 60.0 and 99.2 %, respectively. Of 61 samples that were positive in one, two or three of the assays, 56 were available for discordance analysis. Discordance analysis was performed by culture of toxinogenic strains. The concordance of VIDAS, PTAB and PCR with culture was 53.6 % (30/56), 55.4 % (31/56) and 71.4 % (40/56), respectively. It was concluded that real-time PCR had the highest concordance with toxinogenic culture and is therefore the preferred method for diagnosing CDAD in faecal samples. It was also concluded that diagnosis of patients with diarrhoea who have been hospitalized for more than 72 h should focus mainly on the detection of C. difficile, irrespective of the physician's request.

Clostridium difficile ribotype 027, toxinotype III, the Netherlands.

Outbreaks due to Clostridium difficile polymerase chain reaction (PCR) ribotype 027, toxinotype III, were detected in 7 hospitals in the Netherlands from April 2005 to February 2006. One hospital experienced at the same time a second outbreak due to a toxin A-negative C. difficile PCR ribotype 017 toxinotype VIII strain. The outbreaks are difficult to control.

Prospective multicenter evaluation of a new immunoassay and real-time PCR for rapid diagnosis of Clostridium difficile-associated diarrhea in hospitalized patients.

In a prospective multicenter study, 367 fecal samples from 300 patients with diarrhea were tested for Clostridium difficile-associated diarrhea (CDAD) with a new immunochromatography assay for toxins A and B (ICTAB), a real-time PCR on the toxin B gene, and the cell cytotoxicity assay. Twenty-three (6.2%) of the 367 fecal samples were positive by the cell cytotoxicity assay. With the cell cytotoxicity assay as the "gold standard," the sensitivity, specificity, positive predictive value, and negative predictive value for the ICTAB assay and real-time PCR were 91, 97, 70, and 99%, and 87, 96, 57 and 99%, respectively. In conclusion, both the ICTAB and the real-time PCR can be implemented as rapid screening methods for patients suspected of having CDAD.

Coexistence of multiple PCR-ribotype strains of Clostridium difficile in faecal samples limits epidemiological studies.

Clostridium difficile is an important cause of antibiotic-associated diarrhoea. The simultaneous presence of different strains in individual faecal samples has not yet been established, but is important for epidemiological studies. Recurrences of Clostridium difficile-associated diarrhoea (CDAD) are observed in 15-20 % of patients and have been reported as relapses or reinfections with a new strain. In a period of 1 year, 28 faecal samples from 23 patients with a first episode of CDAD were collected at the Leiden University Medical Centre. In addition, 52 faecal samples from 23 patients, from three different hospitals, with one (n = 19), two (n = 2) or three (n = 2) recurrences were studied. PCR-ribotyping was applied as the standard typing method for the isolates. The toxinogenic and clindamycin-resistance profiles of the isolates was determined by PCR. Of 23 patients with a first episode of CDAD, two (8.7 %) harboured two different types, with no differences in toxinogenicity or clindamycin resistance, within one faecal sample. One of these 23 patients showed two types in three faecal samples from the same episode. Of the 23 patients with recurrences, six (26 %) showed a different strain type isolated in a recurrent episode. The number of cases of multiple C. difficile strains in faecal samples from patients with a first episode of CDAD did not differ significantly from the number of different strains present in recurrent episodes (chi-square test, P < or = 0.2). This observation limits the application of typing methods for studying the epidemiology of CDAD.

Characterization of toxin A-negative, toxin B-positive Clostridium difficile isolates from outbreaks in different countries by amplified fragment length polymorphism and PCR ribotyping.

Clinical Clostridium difficile isolates of patients with diarrhea or pseudomembranous colitis usually produce both toxin A and toxin B, but an increasing number of reports mention infections due to toxin A-negative, toxin B-positive (A(-)/B(+)) strains. Thirty-nine clinical toxin A(-)/B(+) isolates, and 12 other unrelated isolates were obtained from Canada, the United States, Poland, the United Kingdom, France, Japan, and The Netherlands. The isolates were investigated by high-resolution genetic fingerprinting by use of amplified fragment length polymorphism (AFLP) and two well-described PCR ribotyping methods. Furthermore, the toxin profile and clindamycin resistance were determined. Reference strains of C. difficile representing 30 known serogroups were also included in the analysis. AFLP discriminated 29 types among the reference strains, whereas the two PCR ribotyping methods distinguished 25 and 26 types. The discriminatory power of AFLP and PCR ribotyping among 12 different unrelated isolates was similar. Typing of 39 toxin A(-)/B(+) isolates revealed 2 AFLP types and 2 and 3 PCR ribotypes. Of 39 toxin A(-)/B(+) isolates, 37 had PCR ribotype 017/20 and AFLP type 20 (95%). A deletion of 1.8 kb was seen in 38 isolates, and 1 isolate had a deletion of approximately 1.7 kb in the tcdA gene, which encodes toxin A. Clindamycin resistance encoded by the erm(B) gene was found in 33 of 39 toxin A(-)/B(+) isolates, and in 2 of the 12 unrelated isolates (P < 0.001, chi-square test). We conclude that clindamycin-resistant C. difficile toxin A(-)/B(+) strain (PCR ribotype 017/20, AFLP type 20, serogroup F) has a clonal worldwide spread.

Regulation of alpha1,3galactosyltransferase expression in pig endothelial cells. Implications for xenotransplantation.

The disaccharide galactose(alpha)1,3 galactose (the alphaGal epitope) is the major xenoantigen responsible for the hyperacute vascular rejection occurring in pig-to-primates organ transplantation. The synthesis of the alphaGal epitope is catalyzed by the enzyme alpha1,3-galactosyltransferase (alpha1,3GalT). To be able to control porcine alpha1,3GalT gene expression specifically, we have analyzed the upstream portion of the alpha1,3GalT gene, and identified the regulatory sequences. Porcine alpha1,3GalT transcripts were detected by 5' RACE analysis, and the corresponding genomic sequences were isolated from a phage library. The porcine alpha1,3GalT gene consists of at least 10 different exons, four of which contain 5' untranslated sequence. Four distinct promoters, termed A-D, drive alpha1,3GalT gene transcription in porcine cells. A combination of alternative promoter usage and alternative splicing produces a series of transcripts that differ in their 5' portion, but encode the same protein. Promoters A-C have been isolated, and functionally characterized using luciferase reporter gene assays in transfected porcine endothelial cells (PEC-A). Promoter preference in porcine endothelial cells was estimated on the basis of relative transcript levels as determined by real-time quantitative PCR. More than 90% of the alpha1,3GalT transcripts in PEC-A cells originate from promoter B, which has characteristics of a housekeeping gene promoter. While promoter preference remains unchanged, alpha1,3GalT mRNA levels increase by 50% in 12 h upon tumour necrosis factor alpha-activation of PEC-A cells. However, the magnitude of this change induced by inflammatory conditions could be insufficient to affect cell surface alpha1,3-galactosylation.